1. Field of the Invention
Embodiments of the present invention relate to methods and compositions for molecular cloning, particularly, cloning of a donor DNA into an acceptor vector at a predetermined location.
2. Background of the Invention
In molecular biology research and biotechnology industry, there is a constant need of cloning a desired DNA molecule into a vector, preferably at a predetermined location. A conventional method for cloning a donor DNA into an acceptor vector at a predetermined location, such as a plasmid, usually involves six major steps: (i) digesting the acceptor vector DNA with one or two restriction endonucleases and purifying the linearized vector; (ii) treating the linearized vector with Calf Intestinal Phosphatase (CIP) to minimize self-recircularization of the linearized vector without the donor DNA during ligation; (iii) amplifying the donor DNA by a polymerase chain reaction (PCR) using PCR primers that will add to the 5′- and 3′-ends of the amplified donor DNA restriction enzyme recognition site(s) for the one or two restriction endonucleases used to linearize the vector DNA; (iv) digesting the amplified donor DNA with the same restriction endonucleases used to linearize the vector DNA and purifying the digested donor DNA; (v) ligating the purified donor DNA and the purified linearized vector using DNA ligase; and (vi) transforming the ligation products into recipient cells, such as competent Escherichia coli cells, and selecting transformants containing the desired cloning product where the donor DNA is inserted in the vector at the desired cloning site. The conventional cloning method is cumbersome and time-consuming. It has relatively low cloning efficiency. It is also limited by the availability of suitable restriction enzyme recognition sites on the vector and the donor DNA.
Recombination-based methods have been used to expedite cloning. For example, a recombineering-based method for generating conditional knockout mutations was described by Pentao Liu, et al. (Genome Res (2003) 13: 476-484). The method uses a phage-based E. coli homologous recombination system without the need for restriction enzymes or DNA ligases. In particular, the method uses homologous recombination mediated by the λ phage Red proteins, to subclone DNA from bacterial artificial chromosomes (BACs) into high-copy plasmids by gap repair, and together with Cre or Flpe recombinases, to introduce loxP or FRT sites into the subcloned DNA. Longer than 45-55-bp regions of homology are used in the method. Like several other recombination-based methods, the method depends on specific sequences within the acceptor vector and the expression of specific phage proteins in the host cell, thus restricts the user to particular vectors and host cells.
Another example of recombination-based cloning is developed by Clontech Laboratories, Inc. (Mountain View, Calif. 94043), as In-Fusion™ PCR Cloning Kits. The Kits purport to allow cloning of any PCR fragment into any linearized vector in a single step without restriction digestion of the PCR fragment, ligation or blunt-end polishing. The In-Fusion™ system allows to fuse the ends of the PCR fragment to the homologous ends of a linearized vector. The 3′ and 5′ regions of homology are generated by adding 15 bp extensions to both PCR primers that precisely match the ends of the linearized vector. The method consists of 30 minutes incubation of the linearized vector with the PCR fragment and In-Fusion™ enzyme, followed by transformation of E. coli. The In-Fusion™ enzyme is a proprietary protein that converts the double-stranded extensions into single-stranded DNA and fuses these regions to the corresponding ends of the linearized vector. While the In-Fusion™ system does allow rapid directional cloning of PCR product without the restrictions of vectors and host cells, it depends on the proprietary In-Fusion™ enzyme, thus restricts the user to the In-Fusion™ PCR Cloning Kits or similar systems sold by Clontech or its affiliates.
Therefore, there is a need for a novel fast and simple method for cloning a donor DNA in a predetermined location of a vector. Such method is described in the present application. The method according to embodiments of the present invention has all the privileges of the In-Fusion™ PCR Cloning Kits with even higher cloning efficiency using an enzyme cocktail instead of the In-Fusion™ enzyme.